1. Field of the Invention
The invention, in the fields of virology, immunology and medicine, relates to a method for inactivating a pathogenic microorganism or virus and/or enhancing its immunogenicity by treating the microorganism or virus with a polycyclic aromatic hydrocarbon, preferably hypericin or an analog thereof. The invention also provides a method for producing a vaccine, comprising inactivating a microorganism or virus in a way which preserves and enhances its immunogenic properties using the method of the invention, vaccines produced by this method, and methods for vaccinating against a disease, comprising immunization with the vaccine of the present invention.
2. Description of the Background Art
Vaccination against bacterial or viral induced diseases is an old concept which dates back to Louis Pasteur. The first vital vaccine devised by Lister involved the inoculation of humans with cow pox virus (Vaccinia) to obtain immunity against the human pox virus (Variola). Since then, a wide variety of vaccines against numerous childhood diseases such as mumps, measles and German measles (Rubella), as well as against poliovirus and Hepatitis B virus have been developed and approved for use.
The requirements for a vaccine against a disease-causing virus have been defined. Effective vaccine preparations must exhibit several properties described below.
The virus preparation in the vaccine must be rendered non-infectious either by killing the virus or, in the case of live virus vaccines, attenuating the virus. Infectivity is completely abolished by subjecting the virus to processes of inactivation which essentially kill the virus. Often, however, complete inactivation of the virus results in reduced protective capacity. This phenomenon is perhaps best known in polio virus vaccines, wherein the live virus vaccine (the Sabin vaccine) has a superior protective index compared to the killed virus vaccine (the Salk vaccine). However, whereas there are no reports of disease caused by the Salk vaccine, the Sabin vaccine has caused disease in several hundred individuals.
The antigenic properties of the virus in the vaccine preparation must be preserved. Viral antigenic determinants must be available for recognition, processing and presentation by antigen-presenting cells of the immune system of the vaccinated individual. The immunity which is elicited against these antigenic determinants must be capable of reacting with an invading virus to inactivate it before it causes disease. A major goal in vaccine preparation is to enhance the immunogenicity of those antigenic determinants which are important for protective immunity but are often weak immunogens.
This process is most commonly achieved in two ways, often used in combination. First, adjuvants are incorporated into vaccines to enhance non-specifically the immune response against the viral components. Examples for such adjuvants are the pertussis in the Diphtheria-Pertussis-Tetanus (DPT) vaccine, or lipid fractions derived from mycobacteria.
Second, advantage is taken of the knowledge that immune reactions against antigens in particulate form are considerably stronger and more prolonged than responses to soluble antigens. The particulate form slows down the turnover of the antigens and the rate of their degradation. Since clearance from the body is prolonged, the immune system is exposed to more protracted stimulation by the antigen. Because of this consideration, many vaccines are formulated to achieve a particulate form of the antigen. The most common approaches in use involve precipitation of viral components on aluminum hydroxide (alum precipitation).
Modern molecular biology and recombinant DNA technology has also revolutionized the vaccine field. It has enabled the engineering of recombinant and genetically modified viruses from which deleterious disease-inducing genes and other undesirable regulatory genes have been deleted. By such methods, DNA encoding the most important antigens, in particular viral surface antigens, are cloned in bacterial and yeast systems and the antigens obtained therefrom in high yield for immunization. Some recent recombinant vaccine preparations encompass only such selected antigens (Sumner, J. W. et al., Virology 183:703-710 (1991); Gotch, F. M. et al., AIDS 5:317-320 (1991); Taylor, J. et al., J. Virol. 65:4263-4274 (1991). In other approaches, DNA encoding surface proteins from a pathogenic virus is packaged in a "chimeric" non-pathogenic virus (such as Vaccinia), and this new engineered virus serves as the immunizing preparation.
As mentioned above, inactivation of a pathogenic virus, such as a retrovirus, is often an essential step in the preparation of a vaccine. Numerous methods of inactivation are presently in use. These include fixation and cross-linking of the virus with formaldehyde or glutaraldehyde, heat inactivation, or inactivation with heavy metal salts such as cesium chloride and others. Nevertheless, there, is still a well-recognized need for improved means of virus inactivation for production of vaccines with greater efficacy, safety and other desirable qualities.
Hypericin is an aromatic polycyclic dione compound which has previously been found to possess photodynamic properties (Duran et al., Photochem. Photobiol. 43:677-689 (1986); Giese Photochem. Photobiol. Rev. 5:229-255 (1980)). In the presence of light (and possibly other sources of energy), this compound excites oxygen to its singlet state and is capable of generating superoxide radicals which can lead, among other things, to oxidation of tryptophan imidazole groups in proteins and oxidation of fatty acids in biological systems.
The present inventors and their colleagues have investigated the use of hypericin and its quinone-containing analogs in the inactivation of retroviruses. See, for example, U.S. Pat. No. 5,047,435; Degar et al., In: HIV Disease: Pathogenesis and Therapy, University of Miami, 1991, abstract No. 1-16; Lavie et al., Fifth Int'l Conf. on AIDS, Montreal, 1989, abstract C.501; Lavie et al. Proc. Natl. Acad. Sci. USA 86:5963-5967 (1989); Lavie et al. Ann. New York Acad. Sci. USA 616:556-562 (1990); Lavie, G. et al., In: HIV Disease: Pathogenesis and Therapy, University of Miami, 1991, abstract 1-27; Meruelo, D. et al., Proc. Natl. Acad. Sci. USA 85:5230-5234 (1988); Meruelo, D. et al. In: HIV Disease: Pathogenesis and Therapy, University of Miami, 1991, abstract 1-291; Valentine et al., Fifth Int'l Conf. on AIDS, Montreal, 1989, abstract M. C. P. 18; Weiner et al., Fifth Int'l Conf. on AIDS, Montreal, 1989, abstract C-608.